Methods for diagnosing hypertension by detecting a mutation in the human G protein β3 subunit gene

ABSTRACT

A method of diagnosing a disease comprising determining the presence of a genetic modification in a gene obtained from a subject which encodes a human G protein beta3 subunit. Also disclosed is a method for establishing the relative risk of developing a disorder associated with G protein dysregulation.

BACKGROUND OF THE INVENTION:

(i) Field of the Invention

The present invention relates to a method for the diagnosis of diseasesby genetic analysis, in particular the analysis of genes for subunits ofthe human guanine nucleotide-binding proteins (G proteins).

(ii) Description of the Related Art

Heterotrimeric guanine nucleotide-binding proteins (G proteins) have anoutstanding importance in intracellular signal transduction. Theymediate the relaying of extracellular signals after stimulation ofhormone receptors and other receptors which undergo a conformationalchange after receptor activation. This leads to activation of G proteinswhich may subsequently activate or inhibit intracellular effectors (eg.ion channels, enzymes). Heterotrimeric G proteins consist of threesubunits, the α, β and γ subunits. To date, several different αsubunits, 5 β subunits and about 12 γ subunits have been detected bybiochemical and molecular biological methods (Binbaumer, L. andBimnbaumer, M. Signal transduction by G proteins: 1994 edition.J.Recept.Res. 15:213-252, 1995; Offermanns, S. and Schultz, G. Complexinformation processing by the transmembrane signaling system involving Gproteins. Naunyn Schmiedebergs Arch.Pharmacol. 350:329-338, 1994;NÜrnberg, B., Gudermann, T., and Schultz, G. Receptors and G proteins asprimary components of transmembrane signal transduction. Part 2. Gproteins: structure and function. J.Mol.Med. 73:123-132, 1995; Neer, E.J. Heterotrimeric G proteins: Organizers of Transmembrane Signals. Cell80:249-257, 1995;. Rens-Domiano, S. and Hamm, H.E. Structural andfunctional relationships of heterotrimeric G-proteins. FASEB J.9:1059-1066, 1995).

Receptor-medi ated activation of certain α subunits can be inhibited bypretreatment with pertussis toxin (PTX). These include, in particular,the α isoforms αi1, αi2 and αi3, and various oα subunits. G proteins ofthese types are also referred to as PTX-sensitive G proteins.

SUMMARY OF THE INVENTION-

We have found that a genetic modification in the gene for human Gprotein β3 subunits is suitable for the diagnosis of diseases. Thisgenetic modification is particularly suitable for establishing the riskof developing a disorder associated with G protein dysregulation.

The invention furthermore relates to a method for establishing arelative risk of developing disorders associated with G proteindysregulation for a subject, which comprises comparing the gene sequencefor an G protein β3 unit of the subject with the gene sequence SEQ IDNO:1, and, in the event that a thymine (T) is present at position 825,assigning the subject an increased risk of disease.

BRIEF DESCRIPTION OF THE DRAWING:

FIG. 1 depicts a comparison of genes from normotensives andhypertensives by restriction enzyme analysis.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS:

The genetic modification which has been found is located in the gene forhuman G protein β33 subunit. This gene has been described by Levine etal. (Proc. Natl. Acad. Sci USA, 87, (1990) 2329-2333). The coding regionhas an Ser codon (TCC) at position 275, while subjects with an increasedrisk of a disease associated with G protein dysregulation have the codonTCT, which likewise codes for Ser, at this position. The geneticmodification is a base substitution at position 825 in which a cytosine(C) is replaced by thymine (T). However, this base exchange is “silent”at the amino-acid level, ie. it does not lead to incorporation of adifferent amino acid at this position. The sequence found in subjectswith an increased risk of disease is depicted in SEQ ID NO:1 in thesequence listing.

The genetic modification which has been found usually occurs inheterozygous form.

Disorders associated with G protein dysregulation are defined asdiseases in which the G protein is involved in signal transduction anddoes not carry out its function in a physiological manner.

The dysregulation may have a number of causes, for example amodification in the structural gene or modified gene expression.

The disorders include cardiovascular diseases, metabolic disturbancesand immunological diseases.

Cardiovascular diseases which may be mentioned are:

Hypertension, pregnancy hypertension (gestosis, hypertension inpregnancy), coronary heart disease, localized and/or generalizedatherosclerosis, stenoses of blood vessels, restenosis afterrevascularizing procedures (eg. PTCA with and without stentimplantation), tendency to stroke or thrombosis and increased plateletaggregation.

Metabolic disturbances which may be mentioned are:

Metabolic syndrome, insulin resistance and hyperinsulinemia, type IIdiabetes mellitus, diabetic complications (eg. nephropathy, neuropathy,retinopathy, etc.) disturbances of lipid metabolism, disturbances ofcentral chemoreception (CO₂tolerance, acidosis tolerance, sudden infantdeath (SIDS)).

Immunological diseases which may be mentioned are:

Impaired strength of the body's immune response (formation ofimmunoglobulins, aggressiveness of T cells and NK cells), impairedgeneral tendency to proliferation, including wound-healing capacity,tendency to develop tumors and proliferation including metastasizingpotential of malignantly transformed cells, duration of the latencyperiod after HIV infection until the disease becomes clinically evident,Kaposi sarcoma, tendency to cirrhosis of the liver, transplant toleranceand transplant rejection.

The use of the genetic mutation according to the invention isparticularly suitable for establishing the risk of developinghypertension.

The invention furthermore relates to the production of transgenicanimals harboring the genetic mutation described above. Transgenicanimals of this type are of great importance in particular as animalmodels for the investigation and therapy of the disorders describedabove. The methods for generating transgenic animals are generally knownto the skilled worker.

For the method according to the invention for establishing the relativerisk of developing a disease, body material containing the subject'sgenetic information is taken from a subject. This is achieved as a ruleby taking blood and isolating the nucleic acid therefrom.

The structure of the gene for the G protein β3 subunit is establishedfrom the subject's isolated nucleic acid and is compared with thesequence indicated in SEQ ID NO:1.

The structure of the gene can be established by sequencing of thenucleic acid. This can take place either directly from the genomic DNAor after amplification of the nucleic acid, for example by the PCRtechnique.

The structure of the gene can take place at the genomic level or else atthe mRNA or cDNA level.

It is preferably established by sequencing after PCR amplification ofthe cDNA. The primers suitable for the PCR can easily be inferred by theskilled worker from the sequences depicted in SEQ ID NO:1 The procedurefoo this is advantageously such that in each case a primer binding astrand and complementary strand in front of and behind the relevant baseposition 825 is chosen.

However, other methods can also be used for comparison of the genes, forexample selective hybridization or appropriate mapping with restrictionenzymes. The C→T base exchange at the position 825 described. aboveleads to loss of a cleavage site for the restriction enzyme Dsa I, whichis likewise used to detect this genetic polymorphism.

If the subject ha s a thymine (T) at position 825, he is to be assigneda greater risk of disease than a subject with a cytosine (C) at thisposition.

The invention is illustrated further in the following examples.

EXAMPLE 1

Detection of the genetic modification in hypertensives by sequencing

An enhanced susceptibility to activation of PTX-sensitive G proteins wasdetected in preliminary investigations on patients with essentialhypertension. This detection was possible in immortalized cells frompatients having as phenotypical marker an enhanced activity of the Na/Hexchanger. The enhanced susceptibility to activation of PTX-sensitive Gproteins has important consequences for cellular function. These includeenhanced formation of intracellular second messenger molecules (eg.inositol 1,4,5-trisphosphate), enhanced release of intracellular Ca²⁺ions, increased formation of immunoglobulins and an increased rate ofcell growth. Since these changes can be detected in immortalized cellsand after a long duration of cell culturing, it may be assumed that thismodification is genetically fixed (Rosskopf, D., Frömter, E., andSiffert, W. Hypertensive sodium-proton exchanger phenotype persists inimmortalized lymphoblasts from essential hypertensive patients-a cellculture model for human hypertension. J.Clin.Invest. 92:2553-2559, 1993;Rosskopf, D., Hartung, K., Hense, J., and Siffert, W. Enhancedimmunoglobulin formation of immortalized B cells from hypertensivepatients. Hypertension 26:432-435, 1995; Rosskopf, D., Schröder, K.-J.,and Siffert, W. Role of sodium-hydrogen exchange in the proliferation ofimmortalised lymphoblasts from patients with essential hypertension andnormotensive subjects. Cardiovasc.Res. 29:254-259, 1995; Siffert, W.,Rosskopf, D., Moritz, A , Wieland, T., Kaldenberg-Stasch, S., Kettler,N., Hartung, K., Beckmann, S., and Jakobs, K.H. Enhanced G proteinactivation in immortalized lymphoblasts from patients with essentialhypertension. J.Clin.Invest. 96:759-766, 1995).

RNA was prepared by standard methods from immortalized cell lines fromhypertensives and was transcribed into cDNA using reverse transcriptase.Using the polymerase chain reaction (PCR), the cDNA coding for the Gprotein β3 subunit was amplified and sequenced. The followingoligonucleotide primers were employed for the PCR:

5′-TGG GGG AGA TGG AGC AAC TG and

5′-CTG CTG AGT GTG TTC ACT GCC.

Compared with the sequence published by Levine et al. (Levine, M. A.,Smallwood, P. M., Moen, P. T.,Jr., Helman, L.J., and Ahn, T. G.Molecular cloning of β3 subunit, a third form of the G protein β-subunitpolypeptide. Proc. Natl. Acad. Sci. USA 87(6):2329-2333, 1990), thefollowing difference was found in the cDNA from hypertensives' cells:nucleotide 825 cytosine (C) in the region of the coding sequence isreplaced by a thymine (T) (nucleotide 1 corresponds to base A in the ATGstart codon). This base exchange leads to a silent polymorphism, ie. theamino acid encoded by the corresponding base triplet (serine) is notaltered by comparison with the original sequence. The DNA sequence foundis described in SEQ ID NO:1.

EXAMPLE 2

Detection of the genetic modification in hypertensives by restrictionenzyme analysis

The figure depicts a comparison of genes from normotensives andhypertensives by restriction enzyme analysis. In this, the cDNA codingfor β3 from cells from normotensives (NT) and hypertensives (HT), whichhad been amplified by PCR, was subjected to a restriction enzymeanalysis using the enzyme Dsa I. The reaction roducts were fractionatedin an agarose gel, which is depicted in the figure.

The complete restriction of β3 cDNA from normotensive cells afterdigestion with Dsa I is clearly evident from the figure. The cDNA fromhypertensives' cells is only partly cut by Dsa I. Apart from thecleavage products to be expected there is also uncleaved PCR product.Reference fragments (markers) are loaded on the left and right forcomparison of sizes. Four of the five DNA sequences from hypertensivesdepicted here show the base exchange described above and areheterozygous for this modification.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 4 <210> SEQ ID NO: 1 <211>LENGTH: 1517 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 1 atgggggaga tggagcaact gcgtcaggaa gcggagcagc tcaagaagcagattgcagat 60 gccaggaaag cctgtgctga cgttactctg gcagagctgg tgtctggcctagaggtggtg 120 ggacgagtcc agatgcggac gcggcggacg ttaaggggac acctggccaagatttacgcc 180 atgcactggg ccactgattc taagctgctg gtaagtgcct cgcaagatgggaagctgatc 240 gtgtgggaca gctacaccac caacaaggtg cacgccatcc cactgcgctcctcctgggtc 300 atgacctgtg cctatgcccc atcagggaac tttgtggcat gtggggggctggacaacatg 360 tgttccatct acaacctcaa atcccgtgag ggcaatgtca aggtcagccgggagctttct 420 gctcacacag gttatctctc ctgctgccgc ttcctggatg acaacaatattgtgaccagc 480 tcgggggaca ccacgtgtgc cttgtgggac attgagactg ggcagcagaagactgtattt 540 gtgggacaca cgggtgactg catgagcctg gctgtgtctc ctgacttcaatctcttcatt 600 tcgggggcct gtgatgccag tgccaagctc tgggatgtgc gagaggggacctgccgtcag 660 actttcactg gccacgagtc ggacatcaac gccatctgtt tcttccccaatggagaggcc 720 atctgcacgg gctcggatga cgcttcctgc cgcttgtttg acctgcgggcagaccaggag 780 ctgatctgct tctcccacga gagcatcatc tgcggcatca cgtctgtggccttctccctc 840 agtggccgcc tactattcgc tggctacgac gacttcaact gcaatgtctgggactccatg 900 aagtctgagc gtgtgggcat cctctctggc cacgataaca gggtgagctgcctgggagtc 960 acagctgacg ggatggctgt ggccacaggt tcctgggaca gcttcctcaaaatctggaac 1020 tgaggaggct ggagaaaggg aagtggaagg cagtgaacac actcagcagccccctgcccg 1080 accccatctc attcaggtgt tctcttctat attccgggtg ccattcccactaagctttct 1140 cctttgaggg cagtggggag catgggactg tgcctttggg aggcagcatcagggacacag 1200 gggcaaagaa ctgccccatc tcctcccatg gccttccctc cccacagtcctcacagcctc 1260 tcccttaatg agcaaggaca acctgcccct ccccagccct ttgcaggcccagcagacttg 1320 agtctgaggc cccaggccct aggattcctc ccccagagcc actacctttgtccaggcctg 1380 ggtggtatag ggcgtttggc cctgtgacta tggctctggc accactagggtcctggccct 1440 cttcttattc atgctttctc ctttttctac ctttttttct ctcctaagacacctgcaata 1500 aagtgtagca ccctggt 1517 <210> SEQ ID NO: 2 <211> LENGTH:340 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 MetGly Glu Met Glu Gln Leu Arg Gln Glu Ala Glu Gln Leu Lys Lys 1 5 10 15Gln Ile Ala Asp Ala Arg Lys Ala Cys Ala Asp Val Thr Leu Ala Glu 20 25 30Leu Val Ser Gly Leu Glu Val Val Gly Arg Val Gln Met Arg Thr Arg 35 40 45Arg Thr Leu Arg Gly His Leu Ala Lys Ile Tyr Ala Met His Trp Ala 50 55 60Thr Asp Ser Lys Leu Leu Val Ser Ala Ser Gln Asp Gly Lys Leu Ile 65 70 7580 Val Trp Asp Ser Tyr Thr Thr Asn Lys Val His Ala Ile Pro Leu Arg 85 9095 Ser Ser Trp Val Met Thr Cys Ala Tyr Ala Pro Ser Gly Asn Phe Val 100105 110 Ala Cys Gly Gly Leu Asp Asn Met Cys Ser Ile Tyr Asn Leu Lys Ser115 120 125 Arg Glu Gly Asn Val Lys Val Ser Arg Glu Leu Ser Ala His ThrGly 130 135 140 Tyr Leu Ser Cys Cys Arg Phe Leu Asp Asp Asn Asn Ile ValThr Ser 145 150 155 160 Ser Gly Asp Thr Thr Cys Ala Leu Trp Asp Ile GluThr Gly Gln Gln 165 170 175 Lys Thr Val Phe Val Gly His Thr Gly Asp CysMet Ser Leu Ala Val 180 185 190 Ser Pro Asp Phe Asn Leu Phe Ile Ser GlyAla Cys Asp Ala Ser Ala 195 200 205 Lys Leu Trp Asp Val Arg Glu Gly ThrCys Arg Gln Thr Phe Thr Gly 210 215 220 His Glu Ser Asp Ile Asn Ala IleCys Phe Phe Pro Asn Gly Glu Ala 225 230 235 240 Ile Cys Thr Gly Ser AspAsp Ala Ser Cys Arg Leu Phe Asp Leu Arg 245 250 255 Ala Asp Gln Glu LeuIle Cys Phe Ser His Glu Ser Ile Ile Cys Gly 260 265 270 Ile Thr Ser ValAla Phe Ser Leu Ser Gly Arg Leu Leu Phe Ala Gly 275 280 285 Tyr Asp AspPhe Asn Cys Asn Val Trp Asp Ser Met Lys Ser Glu Arg 290 295 300 Val GlyIle Leu Ser Gly His Asp Asn Arg Val Ser Cys Leu Gly Val 305 310 315 320Thr Ala Asp Gly Met Ala Val Ala Thr Gly Ser Trp Asp Ser Phe Leu 325 330335 Lys Ile Trp Asn 340 <210> SEQ ID NO: 3 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3 tgggggagat ggagcaactg20 <210> SEQ ID NO: 4 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <400> SEQUENCE: 4 ctgctgagtg tgttcactgc c 21

We claim:
 1. A method for diagnosing an increased likelihood ofhypertension in a human subject comprising determining the presence of agenetic modification in a gene obtained from said subject which encodesa human G protein β₃ subunit by comparing said gene to the gene sequenceof SEQ ID NO: 1, wherein said genetic modification is a substitution ofcytosine by thymine at position 825 in SEQ ID NO: 1, wherein thepresence of said genetic modification is associated with an increasedlikelihood of hypertension.
 2. The method as claimed in claim 1, whereinthe presence of a genetic modification in the gene obtained from asubject is determined by sequencing.
 3. The method as claimed in claim2, further comprising the step of amplifying the gene obtained from thesubject before sequencing.
 4. The method as claimed in claim 2, whereinat least a portion of the gene obtained from the subject is amplified,said portion including the third nucleotide, of the codon that encodesthe amino acid at position 275 of said human G protein β₃ subunit. 5.The method as claimed in claim 1, wherein the presence of a geneticmodification in the gene obtained from the subject is determined byhybridization.
 6. The method as claimed in claim 1, wherein the presenceof a genetic modification in the gene obtained from the subject isdetermined by cleavage using a restriction enzyme.
 7. The method asclaimed in claim 6, wherein the restriction enzyme is Dsa I.